Today's medical diagnostics have a significant role in the health care system. A recent study indicates demand for in vitro diagnostics in the United States alone is over $9 billion and will grow over seven percent annually through 2005 (Freedonia, 2001). Scientists are constantly trying to develop new methodologies and technologies for monitoring the overall health and disease conditions of patients.
For example, C-reactive protein (CRP) is an acute phase reactant that is measured in order to assess the upper respiratory differential (URD) of patients. The concentration of CRP increases several-fold in response to different types of tissue damage and inflammation (Crockson et al, 1966). CRP is considered to be a prototypic acute phase reactant, synthesized in the liver as part of a coordinated response by hepatocytes to tissue injury or inflammation. Also, results from the 1997 Physicians Health Study have sparked interest in the utility of C-reactive protein (CRP) as being a significant disease indicator, particularly for males aged 40-84 (Luhr and Modi, 2000; Ridker et al, 1997). A rapid and accurate method for measuring CRP is needed to distinguish between allergic responses and viral and bacterial infections, and thereby to avoid unnecessary prescription of antibiotics.
In another example, high levels of immunoglobulin E (IgE) are observed when a person suffers from an allergic reaction. IgE is one of the five classes of immunoglobulins produced by humans (others being, IgA, IgD, IgG and IgM), and the main function of these immunoglobulins is to protect against invading parasites. The antigen-specific IgE interacts with mast cells and eosinophils in its role of protecting the host against the invading parasites (Sutton and Gould, 1993). However, in addition to this beneficial role, the same antibody-cell combination is also responsible for allergic or immediate hypersensitivity reactions such as hay fever, asthma, hives and anaphylaxis. IgE levels increase in serum during these events (Heinrich et al, 1999). IgE-mediated allergies are a significant health problem because of the high prevalence, potential severity, and chronicity of the reaction. Apart from the pollution related allergies, it is estimated that up to 8% of children and 2% of adults have allergic reactions to food (Burks and Sampson, 1993; Jansen et al, 1994). Respiratory diseases and allergies are also growing health concerns in most polluted areas, and it is important for the patient or physician to know whether the respiratory symptoms are caused by a pathogen or by IgE-mediated allergic reaction. Accordingly, immunoassays are needed that quickly and accurately detect the amount of circulating IgE in a patient.
Several immunological methods are commonly used for determining the antigen-antibody interactions. The most common are the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, equilibrium dialysis, sedimentation and biosensor (Biacore). Generally, the affinity of an antibody-antigen interaction can range widely from about 104 to about 1010 M−1. The variability in antigen-antibody affinities is largely determined by imperfect matching of the selected antibody with an antigenic epitope of the target antigen. Moreover, antibody-antigen interactions in the lower portion of the affinity range often do not provide enough sensitivity for accurately measuring the amount of target antigen present in certain types of assays.
Selection of an appropriate antigen and antibody is therefore one of the key factors in developing an accurate and sensitive immunoassay for an analyte or disease marker. The efficacy of an immunoassay depends upon specific recognition of the antigen by antibody and the affinity of an antibody for an antigen is a crucial factor that determines how well the particular antibody preparation will perform in different types of immunoassays (Steward and Lew, 1985). For example, a high affinity antibody is needed in sandwich immunoassay format such as an ELISA. In contrast, the same antibody may not be the best type in a competitive format such as an enzyme immunoassay (EIA). Antibodies of moderate affinity are preferable for use in both EIA and affinity chromatography, because they allow the antigen to be dissociated more easily after formation of antibody-antigen complex.
For example, the IgE analyte is itself an “antibody-like” molecule that could be used in either an EIA or an ELISA format. However, EIA and ELISA (or sandwich) assays often permit detection of an antigen-antibody complex at only one time point; generally such assays cannot be monitored continuously. While EIA is a simpler procedure that requires only one antibody to specifically recognize at Fc or Fab portions of IgE antigen, detection with two antibodies as in the ELISA or sandwich assay provides a more sensitive and accurate assay.
Sandwich assays like the ELISA involve forming a complex between the antigen and two different antibodies where the antibodies recognize the antigen at two different sites. The presence of an antigen is only recorded as present after both antibodies have bound.
While the sandwich assay format is often useful, not all sandwich assays perform optimally. For example, the ELISA method requires one of the antibody preparations to have a detection label such as an enzyme tag. Other detection methods suitable for sandwich assays include calorimetric, fluorometric, chemiluminescent, radiometric, and related methods. Detection labels or tag employed in such methods may sterically interfere with antigen-antibody binding. ELISA results may also differ when solid-phase and solution-phase assays are performed, possibly because the kinetics of such reactions differ (Nygren et al, 1987; Azimzadeh et al, 1992). Moreover, when two antibodies are involved in immune complex formation, suboptimal binding or cross-reactivity by one antibody can undermine the accuracy of the entire immunoassay.
Antigen-antibody affinities can be measured in a single assay under a single set of experimental conditions. However, a single assay will provide very little information about the binding site, the cross-reactivity of the antibody with other antigens and how well two antibodies work together to interact with the target antigen. Accordingly, new methods for effectively selecting the best pairs of antibody preparations for immunoassays are needed.